56 1

DEFECTIVE CD2 PATHWAY T CELL ACTIVATION IN SYSTEMIC LUPUS ERYTHEMATOSUS

DAVID A. FOX, JO ANN MILLARD, JONATHAN TREISMAN, WENDY ZELDES, ALICE BERGMAN, JOEL DEPPER, ROBERT DUNNE, and W. JOSEPH McCUNE

CD2 (‘Ill; sheep erythrocyte receptor) is the surface component of an alternative, antigen-indepen- dent pathway of human T cell activation. The response to certain anti-CD2 antibodies is relatively independent of accessory cell signals and therefore provides a direct measurement of T cell function. The CD2 pathway may be important in the differentiation of thymocytes, on which the expression of CD2 precedes the appearance of the CD3-T cell receptor complex. In view of the im- paired T cell regulation of immune responses in patients with systemic lupus erythematosus (SLE), we examined the activation of peripheral blood lymphocytes by anti- CD2 antibodies in 57 SIX patients and 32 normal control subjects. The CD2 pathway response was lower in the SLE patients (P < 0.0001); 18 of the 57 SLE patients had a lower response than any of the control subjects. The SI,E low-responder patients did not differ from the normal-responder patients in terms of disease activity or use of antiinflammatory and immunosuppres- sive medications. Low responses to anti-CD2 were cor- rected to normal by the coaddition of a submitogenic amount of phorbol myristate acetate (1 ng/ml). In some

From the Division of Rheumatology, Rackham Arthritis Research Unit, Department of Internal Medicine. and the Multipur- pose Arthritis Center, IJniversity of Michigan Medical Center, Ann Arbor.

Supported by the 1,upus Foundation of America. Dr. Pox’s work is supported by an Arthritis Foundation Investigator Award (Michigan chapter), by NIH grant AR-38477, and by the Leyota M. Wilson fund of the University of Michigan Medical School.

David A. Fox, MI); Jo Ann Millard, MSc; Jonathan lreis- man, MD; Wendy Zeldes, 13Sc; Alice Hergman, BSc; Joel Depper, MI); Robert 1)unne. HSc; W. Joseph MeCune, MD.

Address reprint requests to David A. Fox, MD, Division of Rheumatology, R4570 Kresge Eye Institute, University of Michi- gan, Ann Arbor, MI 48109-0531.

Submitted for publication April 24, 1990; accepted in re- vised form November 14, 19W.

low-responder patients, the responses were normalized by the removal of non-T cells. The data indicate that some SLE patients have impaired responses to CD2 pathway activation and that this may reflect intrinsic T cell defects and/or regulatory influences of non-T cells.

The clinical manifestations of systemic lupus erythemdtosus (SLE) are produced, in large part, by the effects of pathogenic autoantibodies. Underlying the B lymphocyte abnormalities in SLE, however, are disturbances in regulatory T cell function. T lym- phopenia is common (1-3), and the proportions of T cells in defined phenotypic/functionaI subsets are fre- quently deranged (2-6). T cell function in SLE patients is deficient in several ways, including proliferative responses (7-lo), production of and response to inter- leukin-2 (IL-2) ( 1 1-17), generation of suppressor cell function (18-22), and responses in the autologous mixed lymphocyte reaction (21,23).

Anti-T cell autoantibodies probably play a ma- jor role in producing these abnormalities. Such anti- bodies are heterogeneous, and may be directed at structures on activated T cells or on subsets required for the generation of suppressor function (24-31). A correlation has been found between the major antilym- phocyte antibody specificity in the sera of individual patients and the concurrent abnormalities in the T cell subsets and functions (20,27,29,31).

It is now realized that multiple T lymphocyte surface structures are involved in the activation of resting T cells. Prominent among these is the T cell antigen receptor complex (CD3-TCR). A distinct structure, which has been termed CD2 (previously known as T11 , and first identified as the T cell receptor for sheep erythrocytes [32]), can also convey activa-

Arthritis and Rheumatism, Vol. 34, No. 5 (May 1991)

562 FOX ET AL

tion signals both to mature T cells and to thymocytes (33,34). Although activation through CD2 was first demonstrated using combinations of anti-CD2 antibod- ies, similar effects have been found using a natural ligand of CD2, termed lymphocyte function- associated antigen 3 (LFA-3; recently designated CD58) (35,36). LFA-3 is an integral membrane com- ponent of cells with which T lymphocytes interact, such as thymic epithelial cells and monocytes (37).

In the course of recent studies of intravenous pulse cyclophosphamide treatment of severe SLE, it was observed that the in vitro proliferative response to anti-CD2 antibodies diminished during treatment and at followup, without a concurrent reduction in other T cell responses (38). It was also apparent that the pretreatment response of the patients' T cells to anti- CD2 antibodies was frequently very low. Since abnor- malities of CD2-mediated T cell activation have not previously been documented in SLE, the present study was undertaken to investigate the integrity of this activation pathway in a large group of SLE patients with various levels of disease activity and severity. The results, when compared with those from the healthy control subjects, indicate that approxi- mately 30% of SLE patients have very low prolifera- tive responses to anti-CD2 antibodies. This low re- sponse cannot be explained by the level of disease activity or by the effects of medications.

PATIENTS AND METHODS Patients and controls. Peripheral blood mononuclear

cells were obtained from 57 patients with SLE (8 men and 49 women) who were receiving followup care at the University of Michigan Medical Center. These patients met the Amer- ican Rheumatism Association 1982 revised criteria for the classification of SLE (39), and they had not been treated with cyclophosphamide. The mean * SD age of the patients was 34 * 11 (range 19-62). Characteristics of these patients, who represented a wide range of disease manifestations and levels of activity, are described below (see Results).

The control group consisted of 32 healthy volunteers (14 men and 18 women), with a mean f SD age of 30 * 7 (range 19-55). In certain experiments, additional mononu- clear cell samples were obtained from SLE patients who had been treated with intravenous cyclophosphamide within the previous 3 months.

Activation of T cells. Mononuclear cells were isolated from heparinized peripheral blood by centrifugation over Ficoll-Hypaque (Pharmacia, Piscataway, NJ), followed by 3 washes in minimum essential medium containing 2% calf serum. For some experiments, T cells (E+) and non-T cells (E-) were separated by rosetting with sheep erythrocytes. Aliquots of 100,000 cells were cultured in RPMI 1640 me-

dium with 10% fetal calf serum in 96-well round-bottom plates in a volume of 0.2 ml/well. Triplicate wells contained either no stimulus or phytohemagglutinin (PHA) (0.5 pg/ml; Burroughs Wellcome, Research Triangle Park, NC), phorbol myristate acetate (PMA) (1 ng/ml; Sigma, St. Louis, MO), a mitogenic combination of 2 anti-CD2 monoclonal antibodies (anti-Tll, and anti-Tll, [see ref. 33]), a mitogenic anti-CD3 antibody (anti-T3B [see ref. 40]), or tetanus toxoid (final concentrations 1:200 and 1:400; Massachusetts Department of Public Health, Boston, MA), or combinations of some of these stimuli. The monoclonal antibodies were generously provided by Drs. Stuart Schlossman and Ellis Reinherz (Dana-Farber Cancer Institute, Boston, MA), and were used at final concentrations of 1500.

Cultures were maintained at 37°C in a humidified incubator with 5% CO,. To detect cell proliferation, 0.8 WCi of tritiated thymidine was added on day 4 (day 6 for tetanus toxoid) and cultured for 18 hours. Cultures were then harvested using a multichannel instrument and counted in a liquid scintillation counter. Background counts per minute from unstimulated cultures were subtracted from the raw counts. For tetanus toxoid, the cpm from the dilution that yielded the higher proliferative response for an individual subject was selected.

Surface marker analysis. A panel of monoclonal antibodies was used to quantitate surface marker expression on mononuclear cells from patients with SLE and from control subjects. Markers assessed included pan-T cell antigens (CD2 and CD3), T cell subset markers (CD4, CD8, and CDw60), T cell activation antigens (CD25 and CD26), HLA-DR, CD20 (a mature B cell antigen), CD56 (a natural killer cell antigen), and CDl lb (expressed on monocytes and a small T cell subset). Antibodies identifying T cell antigens (33,34,4143), except for anti-CDw60 (44), were the gener- ous gift of Drs. Stuart Schlossman and Ellis Reinherz. Other antibodies were generously provided by Drs. Lee Nadler (Dana-Farber Cancer Institute) (45,46), Jerome Ritz (Dana- Farber Cancer Institute) (47), and Robert F. Todd (Univer- sity of Michigan) (48).

For surface marker analysis, aliquots of one million cells were incubated with optimal concentrations of monoclo- nal antibodies in a total volume of 0.2 ml for 30 minutes at 4°C. Following 2 washes, a saturating amount of fluorescein- conjugated goat anti-mouse immunoglobulin (Tago, Burlingame, CA) was added. After 30 minutes at 4"C, the samples were again washed twice, fixed with 1 ml of 1% formalin, and analyzed with an Epics C flow cytometer (Coulter, Hialeah, FL).

Measurement of interleukin-2 receptors (IL-2R). Mononuclear cells from patients with either high or low proliferative responses to antLCD2 monoclonal antibodies (anti-T11, + anti-T1 13) were cultured with these antibodies (1 500) for 72-96 hours in RPMI 1640 medium with 10% fetal calf serum at two million cells per ml. Cultured cells were washed 5 times, and resuspended at a concentration of ten million viable cells per ml in binding medium containing RPMI 1640 with 1% bovine serum albumin, 1 mg/ml of human IgG, 25 mM HEPES, and 0.1% sodium azide. For measurement of total IL-2R, triplicate aliquots of one million cells were incubated with 19 ng of tritiated anti-IL-2R (anti-Tac) monoclonal antibody for 60 minutes at room

IMPAIRED CD2 RESPONSES IN LUPUS PATIENTS 563

temperature in a total volume of 0.2 ml. The mixture was then layered over a 0.75 ml sucrose cushion, centrifuged, and the cell pellet was recovered for liquid scintillation counting. Measurement of high-affinity receptors was done using similar procedures, but in the presence of 10-20 fM IL-2 labeled with lZ5I (Amersham, Arlington Heights, IL) and 20 pM unlabeled IL-2 (Cetus, Emeryville, CA).

Measurement of IL-2 production. Supernatants of cells cultured for 48 hours with various activation stimuli were stored at -8o”C, then added at dilutions of 1:2 to 1:16 to triplicate wells containing 5,000 CTLL-2 cells. Prolifera- tion of the CTLL-2 indicator cells was quantitated by measuring thymidine incorporation following addition of 0.8 pCi of tritiated thymidine per well for the final 6 hours of a 24-hour culture.

Induction of the T11, epitope. Incubation of T cells with anti-T11, for 30 minutes at 4°C induces expression of the T11, epitope (33). To determine if this occurred in T cells from SLE patients, E + cells were incubated with anti-Tll, (IgG2) as described (33), then stained with anti-T1 I, (IgG3) and fluoresceinated y3-specific anti-mouse IgG (The Binding Site, Birmingham, UK) or with control antibodies. Samples were then analyzed by flow cytometry.

Calcium fluxes. T cells from SLE patients were suspended at a concentration of 20 x lo6 cells/ml in culture medium (RPMI 1640 supplemented with 10% fetal calf serum, 1% glutamine, and 1% penicillin/streptomycin). Cells were loaded with the dye indo-1 (Molecular Probes, Eugene, OR), at a concentration of 1.5 p M , and maintained at 37°C for 25 minutes in the dark (49). Cells were washed 3 times and resuspended in culture medium. Aliquots of lo6 cells were kept on ice until they were placed in the flow cyto- meter. The ratio of vio1et:blue fluorescence was determined as a function of cell number; the change in the mean ratio was used as a measure of the extent of flux. Readings were taken at multiple time points after the addition of anti-CD2 antibodies (1:500) or ionomycin (5 pg/ml).

Statistical analysis. Responses of SLE and control T cells were compared using Student’s t-test. Correlations within groups of SLE patients were determined by linear regression. Differences in CD2 responses between subsets of SLE patients were analyzed using the Wilcoxon rank sum test.

RESULTS T cell proliferative responses to a panel of

mitogenic stimuli were measured using mononuclear cells from 57 patients with SLE and 32 control subjects (Figure 1). The mean response to anti-CD2 antibodies in the SLE group was less than 60% of that in the control group, and this difference was highly signifi- cant ( P < 0.0001). This result could not be explained by the higher proportion of males in the control group: The response of the male controls to CD2 activation was 63,128 * 9,931 cpm (mean * SEM), which is not significantly different from that of the female controls (69,283 t 7,573 cpm). Differences in proliferative responses between the SLE and control groups were

Anti-CD2 PHA Anll-CD3 PMA Anti-CD3 Tetanus + PMA toxoid

Figure 1. Proliferative responses of mononuclear cells from pa- tients with systemic lupus erythematosus (SLE) and from healthy controls to various T cell stimuli. Proliferation was measured on day 4 of culture, except for tetanus toxoid, which was measured on day 6. PHA = phytohemagglutinin; PMA = phorbol myristate acetate.

also seen with other stimuli, such as PHA, tetanus toxoid, and anti-CD3 plus PMA, but at a level of significance that was not as great as that seen with the CD2 responses (Figure 1). Anti-CD3 or PMA used separately induced comparable levels of proliferation in the SLE and control groups.

All of the normal subjects had anti-CD2 anti- body responses that were over 20,000 cpm, but 18 of the 57 SLE patients had responses that were lower than 20,000 cpm. This latter group was designated “low-responder,’’ and the remaining SLE patients were designated “normal-responder.” Almost all of the differences in the CD2 responses between the SLE patients and the control group were the result of the low-responder patients, many of whom showed counts that were only barely detectable over background in the presence of anti-CD2 antibodies. These low re- sponses were noted at multiple durations of culture (day 2 to day 6), in the presence of higher concentra- tions of antLCD2 antibodies (1: loo), and when cells were precultured in medium alone for 24-48 hours prior to stimulation (data not shown).

Data from the proliferation studies and from concurrent flow cytometric surface marker quantita- tion were then analyzed to determine whether other immunologic parameters correlated with the magni- tude of the CD2 response, either in normal subjects or in SLE patients. As shown in Table 1 , many of these parameters were significantly different between the SLE and control groups. The SLE group exhibited a marked T lymphopenia, which is consistent with find- ings from previous studies (1-3). The proportion of natural killer cells (CD56-t) among the mononuclear

564 FOX ET AL

Table 1. Correlation of immunologic parameters with CD2 activation responses in patients with systemic lupus erythematosus (SLE) and in normal control subjects*

Correlation with anti-CD2 response

SLE patients Control subjects P P P (n = 57) (n = 32) (SLE vs. control) (SLE) (control)

Proliferative response Anti-CD2 PHA Anti-CD3 PMA Anti-CD3 + PMA Tetanus toxoid

CD3 (T3) CD4 (T4) CD8 (T8) CD2 (T11) CD4:CD8 HLA-DR (Ia) CDllb (Mol) CD20 (Bl) CD56 (HNKla) CDw60 (UM4D4)

CD26 (Tal) CDw60/CD3 CD25/CD3 CD26/CD3

Surface marker

CD25 (IL-2R)

38,967 f 4,099 48,077 f 3,340 7,932 * 1,320

14,686 f 3,100 81,076 f 6,205 10,714 t 2,501

37 f 3 21 f 2 13 f 1 31 t 3

2.1 f 0.2 11 * 1 9 f 1 6 f l 4 f 0.5

11 t I 2 f 0.3 6 5 1

0.38 f 0.04 0.07 f 0.01 0.19 f 0.03

66,590 * 6,004 65,467 5 4,531 10,360 f 5,322 17,801 * 4,547

114,827 t 7,797 23,116 f 2,435

63 f 3 38 f 3 16 t 2 63 f 4

2.9 * 0.3 7 f 1

18 f 2 3 * 0.4

10 f 1 17 & 2 2 f 0.4 8 f 1

0.24 t 0.02 0.03 f 0.001 0.12 t 0.02

<0.0001 <0.005

NS NS

0.001 C0.005

<O.OOol <0.0001

NS <O.OOol <0.05

0.01 <0.0001 <0.005 <O.OOol <0.01

NS NS

<0.05 <0.05 <0.05

- <0.001

NS NS

<0.01 NS

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

- <0.05 CO.05 <0.05

NS NS

NS NS NS NS NS NS NS NS NS NS

<0.001 <0.05

NS <0.001 <0.05

* Values are the mean f SEM cpm (proliferative response) or the mean f SEM percentage of mononuclear cells positive over background fluorescence, as determined by flow cytometric analysis (surface markers). Terms in parentheses are other names that have been used to identify the cell surface markers listed. PHA = phytohemagglutinin; NS = not significant. PMA = phorbol myristate acetate; IL-2R = interleukin-2 receptor.

cell population was also reduced, but the proportion of B lymphocytes and the proportion of Ia+ cells were increased. The calculated percentage of T cells bearing markers of activation (CD25 and CD26) was margin- ally greater in the SLE group. Although the percentage of CDw60+ mononuclear cells was lower among the SLE patient group, the calculated proportion of T cells that were CDw60+ was higher. (CDw60 is a marker of a T cell subset that accumulates in autoimmune lesions [44,501).

The reduced expression of CD2 was especially notable in the SLE group, and the proportion of CD2-t cells was somewhat lower than the proportion of CD3+ cells. This was, in part, due to the reduced intensity of CD2 expression that was seen in cells from many of the SLE subjects, causing some cells within the CD2 peak to fall just below the cutoff for back- ground fluorescence (data not shown). The data, there- fore, do not necessarily indicate the existence of a large CD3+, CD2- T cell subset in the SLE patients.

Furthermore, low-intensity expression of CD2 in SLE patients’ cells did not appear to explain the low proliferative responses to anti-CD2 antibodies, since within the SLE group, no correlation was found be- tween the proportion of cells bearing CD2 (or any other surface marker) and the CD2 activation response (Table 1). In the control group, but not the SLE group, the percentage of mononuclear cells and the calculated proportion of T cells bearing markers of T cell activa- tion (CD25 and CD26) correlated positively with the CD2 response (Table 1). There was correlation of CD2 responses with other proliferative responses, although the relationships within the SLE group were different from those within the control group.

To determine whether the low CD2 pathway responses in SLE patients reflected the presence of excessive numbers of monocytes in culture or the inhibitory effects of monocytes and other non-T cells, proliferation was measured using isolated T cells that had been obtained by rosetting with sheep erythro-

IMPAIRED CD2 RESPONSES IN LUPUS PATIENTS 565

Table 2. Proliferative responses of mononuclear cell subsets from patients with systemic lupus erythematosus (SLE) and from a normal control subject to anti-CD2 and anti-CD3 antibodies*

Anti-TI I, + anti-TI I, Anti-CD3

5 x 104 E+, 5 x 104 E+, 5 x 104 E+, 5 x 104 E+, IOSPBMC 5 x 104 E+ 2.5 x 104 E- 5 x 104 E- 105 PBMC 5 x lo4 E+ 2.5 x I O ~ E - 5 x 104 E-

Normal subject 38,723 30,983 ND 26,066 7,759 ND ND ND SLE patient

Patient I 837 3,791 ND 0 1,447 ND ND ND Patient 2 0 7,409 6,041 0 17,258 5,142 19,699 11,677 Patient 3 0 44,902 37,332 2,722 10,086 6,509 ND ND Patient 4 462 64,870 5 1,565 16,100 3,705 40 21,509 24,692 Patient 5 56,903 45,293 54,545 47,184 47,775 509 18,257 17,921

* Values are the mean cpm in triplicate cultures, as measured on day 4. Data are representative of the 12 subjects studied (2 normal controls and 10 SLE patients). E+ and E- cells were separated by rosetting with sheep erythrocytes. PBMC = peripheral blood mononuclear cells; ND = not done.

cytes. As shown in Table 2, removal of non-T cells from the mononuclear cell population restored to normal the low responses in some, but not all, SLE patients’ cells. Therefore, the deficiencies in CD2 pathway function in low-responder SLE patients may be due to intrinsic T cell abnormalities, as well as the negative effects of non-T cells. Responses to anti-CD3 were lower in the absence of E- cells, since mono- cytes are important for the T cell response to soluble anti-CD3 (51).

Experiments were then performed to determine whether the anti-CD2 response in low-responder SLE subjects could be restored to normal by the addition of a second activation stimulus. In these experiments, 2 types of SLE low responders were studied, including some of the subjects described above, as well as a group of SLE patients whose CD2 responses became low after treatment with intravenous cyclophospha- mide (38). The results did not differ between these groups of low responders, and the findings are there- fore combined as a single group for discussion here.

The findings for 20 SLE low responders were compared with those of 12 SLE normal responders in terms of their response to anti-CD2 antibodies or PMA, or both. For cells from the SLE normal re- sponders, the combination of anti-CD2 and PMA gave

an additive, rather than a synergistic, response (Table 3). Despite also having a somewhat lower response to PMA, cells from the SLE low responders had a synergistic response to anti-CD2 antibodies plus PMA, yielding cpm that were not significantly different from the cpm in the normal-responder group. Of the 20 low responders to anti-CD2, 17 had cpm >20,000 when PMA was included in the culture. Some reconstitution of the CD2 response was also seen when IL-2 (1-16 unitdml), instead of PMA, was added to the medium, although this was generally less effective (data not shown) .

Taken together, the above data suggest that T cells from a subgroup of patients with SLE are not effectively activated by the triggering of CD2, although full activation can be restored by inclusion of a comi- togenic stimulus. Defective responses could poten- tially reflect inadequate initial transmembrane signal- ing, which could, in turn, be due to insufficient induction of the TII, epitope. To determine whether the anti-T11, antibody induced prompt expression of the T11, epitope, SLE patient T cells were incubated with anti-T1 l,, and then stained with anti-TI I , and fluorescein isothiocyanate-conjugated anti-mouse Ig that bound to the anti-Tll,, but not the anti-Tll,, antibody. Prompt expression of T11, (50-100% of T

Table 3. antibodies*

Response of cells from patients with systemic lupus erythematosus (SLE) to anti-CD2

Anti-CD2 PMA Anti-CD2 + PMA

Normal responders (n = 12) 54,719 t 5,805 22,997 t 4,162 80,360 -t 10,454 Low responders (n = 20) 5,508 f 1,251 9,555 ? 1,828 62,740 ? 8,730 P (normal vs. low) <0.0001 <0.05 >0.05

* See Patients and Methods for definitions of responder status. Values are the mean 2 SEM cpm. PMA = phorbol myristate acetate.

FOX ET AL

+ Expl -ion0

+ Exp2-ion0

0 0 2 4 6 8 1 0 12

minutes

Figure 2. Generation of calcium fluxes in T cells from patients with systemic lupus erythematosus (SLE) by antLCD2 antibodies. Cells were loaded with indo-1 and analyzed by flow cytometry. AntLCD2 antibodies or ionomycin (iono) were added at time 0. Values are the change in the mean fluorescence intensity (AMFI) of the vio1et:blue ratio over time. Experiment 1 = cells from a low responder to anti-CD2 antibodies; experiments 2 and 3 = cells from 2 different normal responders (see Patients and Methods for details). Data are representative of 12 experiments (8 with SLE patients and 4 with control subjects).

cells positive within 30 minutes) occurred in all 6 experiments, regardless of the level of the proliferative response to anti-T11, plus anti-Tll, (data not shown). The T11, and Tl l , epitopes were able to generate a transmembrane activation signal in all cases, as dem- onstrated by significant calcium fluxes in T cells from both high- and low-responder SLE patients (Figure 2). Similar fluxes were observed with cells from control subjects (data not shown).

One of the components of T cell activation is induced expression of high-affinity IL-2 receptors that contain 2 distinct subunits (52). Experiments were therefore performed to measure both total and high- affinity IL-2R expression after anti-CD2 activation of T cells from both low- and normal-responder SLE subjects and from normal controls. As shown in Table 4, the SLE low responders tended to have lower expression of high-affinity and total IL-2R than did the other groups, but these differences did not, in general, achieve statistical significance. Considerable heteroge- neity was present within each group. When total or high-affinity IL-2R expression was plotted against the response to anti-CD2 antibodies for all SLE patients whose data are included in Table 4, a significant correlation was not detected (data not shown).

Table 4. Interleukin-2 receptor (IL-2R) expression following CD2 activation in cells from patients with systemic lupus erythematosus (SLE) and from normal control subjects*

High-affinity Total IL-2R IL-2R

SLE low responders 498 f 103 3,701 ? 1,377

SLE normal responders 818 f 421 9,946 t 3,752

Normal controls (n = 3) 10,026 2 1,573 P (SLE low responders NS NS

(n = 6)

(n = 4) 1,388 -+ 842

vs. SLE normal responders)

vs. normal controls) P (SLE low responders NS <0.05

~

* See Patients and Methods for definitions of responder status. Values are expressed as the mean 2 SEM no. of IL-2R. NS = not significant (P > 0.05).

To measure the production of IL-2, superna- tants were collected from cultured SLE mononuclear cells that had been activated with a variety of stimuli. Representative findings are shown in Table 5 . Only low levels of CTLL-2 proliferation occurred in the presence of supernatants from cultures activated with

Table 5. from patients with systemic lupus erythematosus (SLE)*

Interleukin-2 production following CD2 activation in cells

PBMC CTLL-2

Experiment 1 AntLCD2 4,262 38 PMA 185 0 Anti-CD2 + PMA 42,825 540 Anti-CD3 + PMA 37,747 70 1

Anti-CD2 29,493 267 PMA 11,887 138 AntLCD2 + PMA 38,173 3,565 Anti-CD3 + PMA 30,667 3,200

Anti-CD2 5,047 162 PMA 1,004 0 Anti-CD2 + PMA 34,361 8,956 Anti-CD3 + PMA 34,125 4,354

Anti-CD2 15,665 90 PMA 11,129 0 Anti-CD2 + PMA 153,954 2,024 Anti-CD3 + PMA 101,193 1,185

* Values are the mean cpm in triplicate cultures and are repre- sentative of 16 experiments (10 low-responder SLE patients and 6 normal-responder SLE patients; see Patients and Methods for details and definitions of responder status). Experiments 1 and 2 used cells from SLE patients who had received intravenous cyclo- phosphamide within the preceding 3 months; experiments 3 and 4 used cells from SLE patients who had received no cytotoxic drugs during the preceding year. PBMC = peripheral blood mononuclear cells; PMA = phorbol myristate acetate.

Experiment 2

Experiment 3

Experiment 4

IMPAIRED CD2 RESPONSES IN LUPUS PATIENTS 567

anti-CD2 antibodies, even when the anti-CD2 re- sponse was in the normal range (e.g., experiment 2 in Table 5) . For the 6 experiments using supernatants from normal-responder SLE subjects, mean 2 SEM CTLL-2 proliferation was only 137 ? 56 cpm, which was not different from that found for the low- responder subjects. Similar low levels of CTLL-2 proliferation were seen using supernatants from PMA- stimulated cultures. Supernatants of cells cultured

Table 6. CD2 responses in clinical subsets of patients with sys- temic lupus erythematosus

Feature present Feature absent at study, mean at study, mean

Clinical characteristic cpm CPm P

Disease activity 2 or 3 40,151 (28) 35,118 (20) 0.91

Renal disease 39,502 (30) 37,549 (17) 0.96 CNS disease 29,097 (17) 42,966 (31) 0.1 I ESR >50 mm/hour 40,972 (22) 31,327 (22) 0.19

(0-3 scale)

24,521 (15) 45,487 (32) 0.03 37,942 (10) 38,906 (36) 0.96

with anti-CD2 plus PMA generally supported much higher levels of CTLL-2 proliferation than did super- Low complement 35,294 (23) 39,168 (20) 0.75 natants of cells cultured with either stimulus alone. Comparing anti-CD2 plus PMA supernatants with anti- NSAIDs 44,211 (14) 35,969 (31) 0.41

Antimalarials 53,104 (7) 36,707 (38) 0.18 CD2 supernatants, the degree of augmentation was Azathioprine 40,470 (7) 38,211 (39) 0.83 >IO-fold in 11 of 16 experiments (including all in Table * The presence or absence of clinical variables at the time of study 5) , and >3-fo1d in (+ I month) was determined retrospectively by chart review. Not all difference was found between low-responder and information was available for all oatients. and the number reme-

gg2!a Currently taking

Of l6 experiments* no

normal-responder SLE groups. The data described above and in Table 5 were

obtained using a 1:4 dilution of supernatant in the CTLL-2 cultures, but similar patterns were seen with dilutions ranging from 1:2 to 1:16. The data described are for supernatants collected from cultures that did not contain anti-IL-2R antibody: In all experiments, a blocking anti-IL-2R antibody was included in parallel initial cultures to prevent utilization of newly pro- duced IL-2 (anti-TAC, at 1: loo), and the results were virtually the same as those obtained in the absence of this antibody (data not shown). The results do not, therefore, demonstrate distinct patterns of IL-2 secre- tion in SLE low versus SLE normal responders fol- lowing CD2 pathway activation.

Clinical parameters were then analyzed in the SLE patients to determine whether low CD2 re- sponses correlated with the use of specific types of medications, the dosage of corticosteroids, or the clinical status. There was no significant positive or negative correlation between the magnitude of the response to anti-CD2 antibodies and the titer of anti- DNA antibodies, the serum creatinine level, the age of the patients, or the dosage of prednisone. The range of dosages of prednisone taken by the low-responder group was 0-60 mg/day, the same as that in the normal-responder group (except for 1 patient in the normal-responder group who was taking 120 mg/day).

The CD2 responses of the SLE patients were then analyzed according to the presence or absence of the following clinical characteristics: high clinical ac- tivity score, renal disease, central nervous system disease, high erythrocyte sedimentation rate, serositis, cytopenia, hypocomplementemia (CH50, C3, or C4),

sented is shown in parentheses. kNS = central nervous systkm; ESR = erythrocyte sedimentation rate; NSAIDs = nonsteroidal antiinflammatory drugs.

and current use of a nonsteroidal antiinflammatory drug, antimalarial agent, or azathioprine. Responses to anti-CD2 antibodies did not differ between the groups with or without these individual characteristics, ex- cept for serositis, which was associated with a mod- estly lower CD2 response (Table 6). The data indicate that low CD2 responses in a subset of SLE patients are not accounted for by overall disease activity or by use of specific medications.

DISCUSSION In this study, the integrity of CD2-mediated T

cell activation was investigated in the peripheral blood cells of 57 SLE patients and 32 healthy control sub- jects. Significantly lower proliferative responses to a mitogenic pair of antLCD2 antibodies were noted in the SLE patients. The difference between the SLE and control groups could be accounted for by a low- responder subset of SLE patients, which composed -30% of the SLE group. Other T cell responses were also reduced in the SLE group, but the difference was especially prominent for the CD2 response.

Most forms of T cell activation, including stim- ulation with mitogenic lectins or soluble antigen, re- quire substantial accessory cell help, which is usually provided by monocytes. Intact T cell responses there- fore depend upon normal function of both monocytes and T cells. Responses to soluble anti-CD3 also re- quire monocytes, in part to bind the Fc portion of the

FOX ET AL

anti-CD3 antibodies to Fc receptors on the monocyte surface (52), a requirement that can be bypassed by the addition of PMA (53). The anti-CD2 antibodies used in these studies activate normal T cells with little or no need for monocytes (33) and are therefore of special value as a direct probe of T cell function. This may be particularly useful in SLE, a disease in which monocyte function may be abnormal (54).

Multiple abnormalities of surface antigen expression were noted on SLE mononuclear cells, including T lymphopenia and reduced CD2 expres- sion. None of these parameters correlated, however, with the level of CD2 responses, which indicates that defective responses did not exclusively reflect low CD2 expression. In the control group, the level of the CD2 response correlated with the proportion of T cells bearing markers of activation. In contrast, in the SLE group, despite higher proportions of activated cells, no such correlation was seen. This suggests that CD2 responses may be regulated differently in SLE pa- tients, even in those who are normal responders, than in healthy control subjects. Validation of this hypoth- esis will, however, require better understanding of the CD2 activation pathway.

Defective CD2 responses in low-responder SLE patients could be normalized by costimulation with minimally mitogenic doses of the phorbol ester PMA. In this respect, SLE low-responder T cells resembled normal thymocytes, which require 2 signals to proliferate through the CD2 pathway (3435). In the case of thymocytes, anti-CD2 antibodies induced IL-2R expression, but not IL-2 production-proliferation occurred only if IL-2 was exogenously provided (34) or if its synthesis was induced by PMA (55). In the case of SLE low responders, experiments designed to precisely localize the CD2 activation defect to either inadequate transmembrane signaling, IL-2R expres- sion, or IL-2 production did not yield a clear-cut answer. Expression of the T11, epitope and induction of calcium fluxes were normal in SLE low responders. Cells from SLE low responders tended to show lower high-affinity and total IL-2R induction by anti-CD2, but the differences between this group and the SLE normal responders or the healthy controls generally did not reach statistical significance.

Interpretation of IL-2R expression in these ex- periments is complicated by the dependence of the full expression of IL-2R on the secretion of IL-2 (56). Detectable production of IL-2 following CD2 stimula- tion was low in both SLE low responders and SLE normal responders, unless PMA was added. It is

possible that SLE normal responders produce low, but functionally important, amounts of IL-2 that are not detectable by currently available assays, and that inadequate IL-2 production limits proliferation in some of the SLE low responders. Alternatively, SLE normal responders might produce non-IL-2 T cell autocrine growth factors following CD2 activation, to a greater extent than the SLE low responders. The low IL-2 production found in this study closely parallels findings with PHA stimulation of T cells from patients with either active or inactive SLE (57).

Patients with active SLE are deficient in the CD4+, CD45R+ suppressor inducer T cell subset (20), which has been reported to respond particularly well to CD2 stimulation (58). Although this subset was not measured in the present study, it is unlikely that deficiency of these cells would account for the SLE low-responder group, since (a) CD2 responses did not correlate with disease activity, and (b) T cell subsets other than the CD4+, CD45R+ population normally also respond vigorously to CD2 activation, as indi- cated by efficient triggering of helper function for immu- noglobulin production by anti-CD2 antibodies (59).

One potential explanation for low CD2 re- sponses might be the presence of a (non-T) cell subset within the mononuclear cell population that sup- presses the CD2 response in the low-responder indi- viduals. In some but not all low responders, purifica- tion of T cells prior to culture significantly enhanced the CD2 response (Table 2). Taken together, the data suggest that the defective responses to anti-CD2 anti- bodies in the SLE low-responder individuals may represent an intrinsic T cell defect (or heterogeneous group of defects), the regulatory effects of non-T cells, and possibly, abnormal T cell responses to such reg- ulatory signals. Low responses do not appear to be the result of SLE-induced organ failure or administration of corticosteroids. Because initial signaling events (as judged by calcium fluxes in the presence of anti-CD2 antibodies) appear to be intact, any intrinsic T cell defects may lie at later stages of the T cell activation process. Whether such defects relate in any way to the pathogenesis of SLE will require further investigation. It is certainly possible that similar abnormalities could be present in other autoimmune diseases, and that such abnormalities could be a secondary consequence of autoimmune processes.

A role for the CD2 pathway has been postulated in the selection of T cells in the human thymus, since in vitro triggering of the thymocyte antigen receptor complex could be shown to provide a negative signal

IMPAIRED CD2 RESPONSES IN LUPUS PATIENTS 569

under conditions in which CD2 triggering provided a positive signal (55,60). Based on these findings, it was hypothesized that thymocytes not subject to negative selection (cells lacking high-affinity autoreactivity) could expand and differentiate by activation through CD2 (60), particularly since a functional ligand of CD2 has been shown to be present in the thymus (36,37). If responses to CD2 in low-responder individuals were defective throughout T cell ontogeny , including the thymocyte stage, a potential consequence could be alterations of the thymic selection process that might predispose to the later development of autoimmune disease. No data that directly support this hypothesis are currently available, however.

It is intriguing that in SLE normal-responder subjects, intravenous cyclophosphamide treatment frequently leads to conversion to a CD2 low-responder state, despite preservation of other T cell proliferative responses (38). This indicates that even in SLE normal responders, the CD2 pathway is uniquely vulnerable and can be regulated in ways distinct from those of other T cell responses. (No information, however, is available regarding the effects of cyclophosphamide on CD2 responses in individuals without autoimmune disease.) Both constitutive and cyclophosphamide- induced CD2 low responsiveness were normalized by small concentrations of the comitogen PMA. This raises the possibility that signal transduction events normally culminating in the activation of protein ki- nase C were defective in these low responders, and that PMA bypassed the defective signaling step(s). Alternatively, the augmenting effect of PMA could reflect modulation of the expression of various T cell surface structures and/or effects on the expression of genes essential to T cell activation by mechanisms other than activation of protein kinase C. Additional work will be necessary to resolve these issues and to precisely identify the mechanisms of abnormal activa- tion through CD2 in patients with SLE.

ACKNOWLEDGMENTS We thank Patricia Bohlke and Sheila Chang for

technical assistance, Clare Rogers for performing flow cy- tometry, and Nancy Forster and Susan Brushingham for preparation of the manuscript. We are grateful to Drs. Stuart Schlossman and Ellis Reinherz for the generous gifts of monoclonal antibodies.

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